1. Field of the Invention
This invention pertains to the field of assays for detecting Bacillus anthracis, the causative agent of anthrax.
2. Background
Anthrax spores were first produced as weapons in the 1950s. Several countries including the former Soviet Union, the United States and Iraq are known to have produced anthrax weapons. Anthrax is a particularly fearsome biological warfare agent, not only because of its deadliness, but also because anthrax weapons are relatively easy to produce and deliver. Production of anthrax spores requires little more than basic laboratory equipment and growth media. Anthrax weapons are comprised of an anthrax source and an industrial sprayer that can produce aerosol particles of the appropriate size for victims to inhale. Such sprayers, for instance, can be mounted on low flying airplanes or other vehicles and used to spread anthrax over a wide area. Because of the ease and relatively small expense involved in producing and delivering anthrax weapons, such weapons are potentially highly attractive weapons of mass destruction for terrorist groups. Thus, in addition to potential organized military conflicts that may give rise to the use of such weapons, terrorist organizations are a potential threat for the use of such weapons in airports, office buildings and other centers of human activity.
Anthrax is caused by Bacillus anthracis, a gram-positive, sporulating bacillus. B. anthracis is a soil bacterium and is distributed worldwide. The organism exists in the infected host as a vegetative bacillus and in the environment as a spore. The anthrax spore is typically the infective form of the bacteria life cycle. Anthrax spores can survive adverse environmental conditions and can remain viable for decades. Animals such as cattle, sheep, goats and horses can contract the spores while grazing. Humans can contract anthrax from inoculation of minor skin lesions with spores from infected animals, their hides, wool or other products, such as infected meat (Franz et al. (1997) J. Am. Med. Assoc. 278(5): 399-411).
The typical mode of entry of the anthrax spore into the body, inhalation, results in an illness known as woolsorter""s disease. After deposit in the lower respiratory tract, spores are phagocytized by tissue macrophages and transported to hilar and mediastinal lymph nodes. The spores germinate into vegetative bacilli, producing a necrotizing hemorrhagic mediastinitis (Franz et al., supra). Symptoms include fever, malaise and fatigue, which can easily be confused with the flu. The disease may progress to an abrupt onset of severe respiratory distress with dyspnea, stridor, diaphoresis and cyanosis. Death usually follows within 24 to 36 hours.
Because the effects of exposure to anthrax are not immediate, and because the initial symptoms are easily confused with the flu, there is a need for a fast method to detect B. anthracis in an environment where B. anthracis may have been released. This need is enhanced by the increasing number of anthrax threats that are called into governmental authorities each year. A fast method for determining whether public places have been exposed to anthrax spores in therefore essential.
Anthrax spores have S-layers, as do spores of many other archea and bacteria. Most S-layers are comprised of repeats of a single protein (Etienne-Toumelin et al., J. Bacteriol. 177:614-20 (1995)). The S-layer of B. anthracis, however, is comprised of at least two proteins: EA1 (Mesnage et al., Molec. Microbiol. 23:1147-55 (1997)) and surface array protein (SAP) (see Etienne-Toumelin, et al., supra). Fully virulent B. anthracis isolates are encapsulated by a capsule that encompasses the S-layer of the bacteria and prevents access of antibodies to both EA1 and SAP (Mesnage et al., J. Bacteriol. 180:52-58 (1998)).
Several methods for detecting B. anthracis have been reported, although none are optimal for quick and reliable detection of anthrax contamination. Detection methods include those based on amplification of nucleic acids that are specific for B. anthracis (Lee, J. Appl. Microbiol. 87:218-23 (1999); Patra, G., FEMS Immunol. Med. Microbiol. 15:223-31 (1996); Ramisse et al, FEMS Microbiol. Lett. 145(1):9-15 (1996); Bruno and Keil, Biosens. Bioelectron. 14:457-64 (1999); and Japanese Patent Nos. 11004693; 6261759; 6253847; and 6253846). The need to conduct time-consuming laboratory procedures to use these amplification methods limits their usefulness for quick identification of anthrax contamination. Other detection methods involve detecting spore-based epitopes of B. anthracis using antibodies (Yu, H., J. Immunol. Methods 218:1-8 (1998); Phillips et al., J. Appl. Bacteriol. 64:47-55 (1988); Phillips et al., FEMS Microbiol. Immunol. 1:169-78 (1988)). Other reported detection methods include an enzyme-linked lectinosorbent assay (Graham et al., Eur. J. Clin. Microbiol. 3:210-2 (1984)) and a method using DNA aptamers that bind anthrax spores (Bruno et al., Biosens Bioelectron. 14(5):457-64 (1999)).
Previous antibody-based detection methods for B. anthracis employed antibodies raised against whole anthrax spores. Such immunogens lead to the production of antibodies that cross-react with other related bacterial species. Longchamps et al., for instance, found that no antibody analyzed in their study was completely specific in recognizing anthrax spores (J. Applied Microbiology 87:246-49 (1999)). At least one study has shown that polyclonal antibodies raised against B. anthracis whole spore suspensions do not react with SAP protein (Mesnage et al, Molec. Microbial. 23:1147-55 (1997)). Closely related bacteria that may cross react with non-specific antibodies include B. cereus, B. thuringiensis and B. mycoides (Longchamp et al., supra.; Phillips et al., FEMS Microbiol. Immunol. 47:169-78 (1988)). This high degree of cross-reactivity is highly problematic for detection of anthrax because these non-toxic cross-reactive strains are widespread. B. thuringiensis in particular is commonly found in the soil, in part because the bacteria is sprayed on crops for its insecticidal qualities.
Therefore, a need exists for improved methods for detecting Bacillus anthracis in the environment. Such methods should be not only provide rapid results, but also should have little or no cross-reactivity with related species that are prevalent in nature. The present invention fulfills this and other needs.
The present invention provides novel methods of detecting Bacillus anthracis. The methods involve contacting a test sample with a capture reagent that can bind to B. anthracis surface array protein (SAP), wherein the capture reagent forms a complex with SAP if SAP is present in the test sample, and detecting whether SAP is bound to the capture reagent. The capture reagent, for instance, can form a complex with the surface array protein if the surface array protein is present in the sample. Presence of the surface array protein is indicative of the presence of B. anthracis in the sample. In one embodiment, SAP comprises a polypeptide with the amino acid sequence shown in SEQ ID NO:1. In another embodiment, the B. anthracis strain is encapsulated.
The capture reagent can comprise an antibody that binds to SAP. In some embodiments, the antibody can be a recombinant antibody, such as a recombinant polyclonal or monoclonal antibody.
In a preferred embodiment, the test sample is collected from a site of suspected or threatened anthrax contamination. In another preferred embodiment, the test sample is collected using a cyclonic device. The test sample does not need to be cultured prior to contacting with the capture reagent.
In some methods of the invention, the capture reagent can be immobilized on a solid surface, such as a microtiter dish. The capture reagent can be immobilized on the solid support prior to contacting the capture reagent with the test sample.
In presently preferred embodiments, the assay methods of the invention are highly sensitive. For instance, in one embodiment, antibodies of the invention used according to the methods of the invention can detect B. anthracis at concentrations at least as low as 10,000 cfu/ml. In a more preferred embodiment, the methods of the invention are capable of detecting B. anthracis at concentrations at least as low as 5,000 cfu/ml. In still more preferred embodiments, the methods of the invention are capable of detecting B. anthracis at concentrations at least as low as 1,800 cfu/ml.
In some embodiments, SAP is detected by contacting SAP with a detection reagent that can bind SAP. Like the capture reagent, the detection reagent can be an antibody that binds SAP. For instance, the detection reagent can bind a different epitope of SAP than the capture agent binds. In some embodiments, the detection reagent comprises a detectable label. The detectable label can be, for instance, a radioactive label, a fluorophore, a dye, an enzyme or a chemilumunescent label.
The invention also provides devices and kits for detecting B. anthracis. The kits typically include, inter alia, a solid support upon which is immobilized a capture reagent which binds to a SAP of B. anthracis, and a detection reagent which binds to the SAP. In some embodiments the solid support is a microtiter dish. In another embodiment, the capture reagent is an antibody, such as a recombinant polyclonal or monoclonal antibody or mixtures thereof. The kit can also include written instructions for using the kit to determine whether a test sample contains B. anthracis. In some embodiments, the kit also comprises a positive control that comprises a polypeptide that comprises an antigenic determinant of B. anthracis SAP. The SAP can be, for example, the amino acid sequence displayed in SEQ ID NO:1.
The invention also provides for recombinant polyclonal antibody preparations that specifically bind to an antigenic determinant of B. anthracis SAP. For instance, the SAP polypeptide can be the amino acid sequence displayed in SEQ ID NO:1.
The phrase xe2x80x9ccapture reagentxe2x80x9d refers to a molecule that specifically binds to a specific target molecule. For instance, the target molecule can be a surface array protein (SAP) of Bacillus anthracis, or a portion thereof. Capture reagents include antibodies as well as naturally and non-naturally-occurring molecules that can specifically bind a target molecule. For instance, peptides that specifically bind a target molecule and are developed using phage display or other combinatorial system are encompassed by this definition.
A xe2x80x9ctest samplexe2x80x9d is a sample obtained from a non-laboratory source that is not known to contain B. anthracis. For example, a sample grown on laboratory growth media or purified from laboratory growth media is not a test sample unless it is not known whether the sample contains B. anthracis. 
The phrases xe2x80x9cspecifically binds toxe2x80x9d or xe2x80x9cspecifically immnunoreactive withxe2x80x9d, when referring to an antibody or other binding moiety refers to a binding reaction which is determinative of the presence of a target antigen in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated assay conditions, the specified binding moieties bind preferentially to a particular target antigen and do not bind in a significant amount to other components present in a test sample. Specific binding to a target antigen under such conditions may require a binding moiety that is selected for its specificity for a particular target antigen. A variety of immunoassay formats may be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an antigen. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactive. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background. Specific binding between an antibody or other binding agent and an antigen generally means a binding affinity of at least 106 Mxe2x88x921. Preferred binding agents bind with affinities of at least about 107 Mxe2x88x921, and preferably 108 Mxe2x88x921 to 109 Mxe2x88x921 or 1010 Mxe2x88x921.
The term xe2x80x9cepitopexe2x80x9d means an antigenic determinant that is capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. Epitopes can include non-contiguous amino acids, as well as contiguous amino acids.
The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one xe2x80x9clightxe2x80x9d (about 25 kDa) and one xe2x80x9cheavyxe2x80x9d chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody""s isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a xe2x80x9cJxe2x80x9d region of about 12 or more amino acids, with the heavy chain also including a xe2x80x9cDxe2x80x9d region of about 10 more amino acids. (See generally, Fundamental Immunology (See, e.g., Paul, Fundamental Immunology, 3rd Ed., 1993, Raven Press, New York).
The variable regions of each light/heavy chain pair form the antibody binding site. The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarily determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. CDR and FR residues are delineated according to the standard sequence definition of Kabat et al., supra. An alternative structural definition has been proposed by Chothia et al. (1987) J. Mol. Biol. 196: 901-917; (1989) Nature 342: 878-883; and (1989) J. Mol. Biol. 186: 651-663.
The term xe2x80x9cantibodyxe2x80x9d is used to mean whole antibodies and binding fragments thereof. Binding fragments include single chain fragments, Fv fragments and Fab fragments The term Fab fragment is sometimes used in the art to mean the binding fragments and Fab fragments from papain cleavage of an intact antibody. The terms Fabxe2x80x2 and F(abxe2x80x2)2 are sometimes used in the art to refer to binding fragments of intact antibodies generated by pepsin cleavage. Here, xe2x80x9cFabxe2x80x9d is used to refer generically to double chain binding fragments of intact antibodies having at least substantially complete light and heavy chain variable domains sufficient for antigen-specific bindings, and parts of the light and heavy chain constant regions sufficient to maintain association of the light and heavy chains. Usually, Fab fragments are formed by complexing a full-length or substantially full-length light chain with a heavy chain comprising the variable domain and at least the CH1 domain of the constant region.
An xe2x80x9cisolatedxe2x80x9d species or population of species means an object species (e.g., binding polypeptides of the invention) that is the predominant species present (i.e., on a molar basis it is more abundant than other species in the composition). Preferably, an isolated species comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods).
The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids, refers to two or more sequences or subsequences that have at least 80%, preferably 85%, most preferably 90-95% nucleotide identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For amino acid sequences, xe2x80x9csubstantially identicalxe2x80x9d refers to two or more sequences or subsequences that have at least 60% identity, preferably 75% identity, and more preferably 90-95% identify, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the nucleic acid or amino acid sequences that is at least about 10 residues in length, more preferably over a region of at least about 20 residues, and most preferably the sequences are substantially identical over at least about 100 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the specified regions (e.g., coding regions).
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat""l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1995 Supplement) (Ausubel)).
Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always  greater than 0) and N (penalty score for mismatching residues; always  less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
A further indication that two nucleic acids or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
xe2x80x9cConservatively modified variationsxe2x80x9d of a particular polynucleotide sequence refers to those polynucleotides that encode identical or essentially identical amino acid sequences, or where the polynucleotide does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent substitutionsxe2x80x9d or xe2x80x9csilent variations,xe2x80x9d which are one species of xe2x80x9cconservatively modified variations.xe2x80x9d Every polynucleotide sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. Thus, silent substitutions are an implied feature of every nucleic acid sequence which encodes an amino acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine and UGG, the only codon for tryptophan) can be modified to yield a functionally identical molecule by standard techniques. In some embodiments, the nucleotide sequences that encode the enzymes are preferably optimized for expression in a particular host cell (e.g., yeast, mammalian, plant, fungal, and the like) used to produce the enzymes.
Similarly, xe2x80x9cconservative amino acid substitutions,xe2x80x9d in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties are also readily identified as being highly similar to a particular amino acid sequence, or to a particular nucleic acid sequence which encodes an amino acid. Such conservatively substituted variations of any particular sequence are a feature of the present invention. Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are xe2x80x9cconservatively modified variationsxe2x80x9d where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. See, e.g., Creighton (1984) Proteins, W.H. Freeman and Company.
The present invention provides novel kits and methods for detecting the presence or absence of B. anthracis in a test sample. The kits and methods are a rapid, accurate and cost-effective means for detecting B. anthracis. The methods involve, in presently preferred embodiments, contacting a test sample with a capture reagent that can bind to B. anthracis SAP. The capture reagent then forms a complex with the SAP if it is present in the sample. The SAP is then detected to determine whether the test sample contains B. anthracis. Typically, detection is accomplished using a detection reagent that specifically binds to B. anthracis SAP. Both capture reagents and the detection reagents typically use binding moieties that can bind to SAP.
Unlike previously available anthrax detection methods, the methods and kits of the invention are highly sensitive. The assays and kits, in presently preferred embodiments, can detect B. anthracis when present in a sample at a concentration of about 104 cfu/ml or less. Preferably, the detection limit for B. anthracis will be about 5xc3x97103 cfu/ml or less, more preferably about 1.8xc3x97103 cfu/ml or less, and still more preferably about 103 cfu/ml or less.
Moreover, the methods and kits are highly specific for B. anthracis. Unlike previously available methods, the methods and kits of the present invention do not suffer from cross-reactivity with non-anthrax microorganisms. Previous methods of detecting B. anthracis relied on antibodies raised against whole anthrax spores, so these assays suffer from significant cross-reactivity. In contrast, the assays of the present invention use binding reagents that are directed to a B. anthracis antigen that is specific for B. anthracis. This antigen is secreted and can be deposited on the surface of anthrax spores and other particles, for example, during the preparation of anthrax-based biological weapons. Thus, in addition to the high specificity of the detection methods of the invention, the methods are more efficient and easy to use because there is no need to disrupt the anthrax spores for binding reagents to bind their antigens. Nor must samples suspected of containing B. anthracis be cultured prior to testing.
A. Binding Moieties that Specifically Bind B. anthracis Surface Array Protein
The assays of the invention involve detecting the presence in a test sample of a B. anthracis SAP polypeptide, which is an antigen that is specific for B. anthracis. The assays for detecting SAP are, in some embodiments, binding assays. In these assays, which include immunoassays, SAP is immobilized on a solid support using a capture reagent that can specifically bind to SAP. The immobilized SAP is then detected using detection reagents that also are capable of specifically binding to SAP. The detection reagents typically include at least a binding moiety and a detectable label.
The invention provides binding reagents that are capable of specifically binding to the SAP antigen. These binding reagents can be used in one or more steps of the assay. For example, the binding reagents can be immobilized on a solid support and used to immobilize SAP on the solid support; such immobilized binding reagents are referred to herein as xe2x80x9ccapture reagents.xe2x80x9d Binding reagents can also be used to detect B. anthracis antigens by, for example, attaching a detectable label to a binding moiety that binds to SAP. Suitable binding moieties include any molecule that is capable of specifically binding to SAP. Antibodies and fragments thereof are examples of binding components that are suitable for use in detection moieties.
1. Types of Binding Moieties
The invention provides binding moieties (or reagents) that can specifically bind B. anthracis SAP polypeptides. Binding reagents can also be, for example, antibodies prepared using as immunogens natural, recombinant or synthetic polypeptides derived from B. anthracis SAP. The amino acid sequence of a B. anthracis SAP is shown as SEQ ID NO:1. Such polypeptides can function as immunogens that can be used for the production of monoclonal or polyclonal antibodies. Immunogenic peptides derived from SAP can also be used as immunogens; such peptides are sometimes conjugated to a carrier polypeptide prior to inoculation. Naturally occurring, recombinantly produced, or synthetic peptides or polypeptides are suitable for use as imnmunogens. These can be used in either pure or impure form. Production of antibodies against SAP polypeptides of the invention is discussed in more detail below. Suitable binding moieties also include those that are obtained using methods such as phage display.
Various procedures known in the art can be used for the production of antibodies that specifically bind to SAP. For the production of polyclonal antibodies, one can use SAP to inoculate any of various host animals, including but not limited to rabbits, mice, rats, sheep, goats, and the like. The SAP polypeptide can be prepared by recombinant means as described above using an expression vector containing a nucleic acid that encodes the B. anthracis SAP. For example, a nucleotide sequence encoding a B. anthracis SAP beginning at approximately 30 amino acids from the published N-terminus (i.e., at the presumed cleavage sequence) is presented in SEQ ID NO:2.
Monoclonal antibodies can be prepared by any technique that provides for the production of antibody molecules by continuous cell lines in culture, including the hybridoma technique originally developed by Kohler and Milstein ((1975) Nature 256: 495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al. (1983) Immunology Today 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Monoclonal antibodies also can be produced in germ-free animals as was described in PCT/US89/02545 (Publication No. WO8912690, published Dec. 12, 1989) and U.S. Pat. No. 5,091,512.
Fragments of antibodies are also useful as binding moieties. While various antibody fragments can be obtained by the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term xe2x80x9cantibody,xe2x80x9d as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv). Single chain antibodies are also useful to construct detection moieties. Methods for producing single chain antibodies were described in, for example, U.S. Pat. No. 4,946,778. Techniques for the construction of Fab expression libraries were described by Huse et al. (1989) Science 246: 1275-1281; these techniques facilitate rapid identification of monoclonal Fab fragments with the desired specificity for SAP. Suitable binding moieties also include those that are obtained using methods such as phage display.
To prepare a suitable antigen preparation, one can prepare an expression library from B. anthracis and screen the library with a polyclonal antibody that is raised against a crude preparation of SAP. The inserts from those expression plasmids that express the SAP are then subcloned and sequenced. The SAP-encoding inserts are cloned into an expression vector and used to transform E. coli or other suitable host cells. The resulting preparation of recombinant SAP is then used to inoculate an animal, e.g., a mouse.
In preferred embodiments, the binding reagents are recombinantly produced polyclonal or monoclonal antibodies that bind to SAP. Recombinant antibodies are typically produced by immunizing an animal with SAP, obtaining RNA from the spleen or other antibody-expressing tissue of the animal, making cDNA, amplifying the variable domains of the heavy and light imnmunoglobulin chains, cloning the amplified DNA into a phage display vector, infecting E. coli, expressing the phage display library, and selecting those library members that express an antibody that binds to SAP. Methods suitable for carrying out each of these steps are described in, for example U.S. patent application Ser. No. 08/835,159, filed Apr. 4, 1997. In preferred embodiments, the antibody or other binding peptides are expressed on the cell surface of a replicable genetic unit, such as a filamentous phage, and especially phage M13, Fd and F1. Most work has inserted libraries encoding polypeptides to be displayed with either pIII or pVIII of these phage, forming a fusion protein which is displayed on the surface of the phage. See, e.g., Dower, WO 91/19818; Devlin, WO 91/18989; MacCafferty, WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO 92/18619 (gene VIII).
In a preferred embodiment, the genes that encode the heavy and light chains of antibodies present in the cDNA library are amplified using a set of primers that can amplify substantially all of the different heavy and light chains. The resulting amplified fragments that result from the amplification step are pooled and subjected to asymmetric PCR so that only one strand (e.g., the antisense strand) is amplified. The single strand products are phosphorylated, annealed to a single-stranded uracil template (e.g., the vector BS45, described in U.S. patent application Ser. No. 08/835,159, which has coding regions for the constant regions of mouse heavy and light chains), and introduced into a uracil DNA glycosylase+ host cell to enrich for vectors that contain the coding sequences for heavy and light chain variable domains.
To screen for phage that express an antibody that binds to SAP, one can attach a label to SAP using methods known to those of skill in the art. In a preferred embodiment, the phage that display such antibodies are selected using SAP to which is attached an immobilizable tag, e.g., biotin. The phage are contacted with the biotinylated antigen, after which the phage are selected by contacting the resulting complex with avidin attached to a magnetic latex bead or other solid support. The selected phage are then plated, and may be screened with SAP to which is attached a detectable label.
In a preferred embodiment, the library is enriched for those phage that display more than one antibody that binds to SAP. Methods and vectors that are useful for this enrichment are described in U.S. patent application Ser. No. 08/835,159. The panning can be repeated one or more times to enhance the specificity and sensitivity of the resulting antibodies. Preferably, panning is continued until the percentage of functional positives is at least about 70%, more preferably at least about 80%, and most preferably at least about 90%.
A recombinant anti-SAP monoclonal antibody can then be selected by amplifying antibody-encoding DNA from individual plaques, cloning the amplified DNA into an expression vector, and expressing the antibody in a suitable host cell (e.g., E. coli). The antibodies are then tested for ability to bind SAP.
Recombinant polyclonal antibodies are particularly preferred because of the various forms of SAP that may be found in clinical samples due to, for example, proteolysis. The diverse fine binding specificity of members of a population of polyclonal antibodies often allows the population to bind to several forms of SAP (e.g., species variants, escape mutant forms, proteolytic fragments) to which a monoclonal reagent may be unable to bind. Methods for producing recombinant polyclonal antibodies are described in U.S. patent application Ser. No. 08/835,159, filed Apr. 4, 1997. Specific methods of producing recombinant polyclonal antibodies that bind to SAP are described in the Examples below.
Polyclonal antibodies can be prepared as described above, except that an individual antibody is not selected. Rather, the pool of phage is used for the screening, preferably using an equal number of phage from each sample. In preferred embodiments, the phage are enriched for those that display more than one copy of the respective antibodies. The phage are then selected for those that bind to SAP. For example, one can use a biotinylated anti-SAP monoclonal antibody and SAP to concentrate those phage that express antibodies that bind to SAP. The biotinylated monoclonal antibody is immobilized on a solid support (e.g., magnetic latex) to which is attached avidin. The phage that are bound to the immobilized SAP are eluted, plated, and the panning repeated until the desired percentage of functional positives is obtained.
2. Detection Reagents of the Invention
The presence of SAP is generally detected using a detection reagent that is composed of a binding moiety that specifically binds to SAP. Suitable binding moieties are discussed above. The detection reagents are either directly labeled, i.e., comprise or react to produce a detectable label, or are indirectly labeled, i.e., bind to a molecule that is itself labeled with a detectable label. Labels can be directly attached to or incorporated into the detection reagent by chemical or recombinant methods.
In one embodiment, a label is coupled to a molecule, such as an antibody that specifically binds to SAP, through a chemical linker. Linker domains are typically polypeptide sequences, such as poly-gly sequences of between about 5 and 200 amino acids. In some embodiments, proline residues are incorporated into the linker to prevent the formation of significant secondary structural elements by the linker. Preferred linkers are often flexible amino acid subsequences that are synthesized as part of a recombinant fusion protein comprising the RNA recognition domain. In one embodiment, the flexible linker is an amino acid subsequence that includes a proline, such as Gly(x)-Pro-Gly(x) (SEQ ID NO:5) where x is a number between about 3 and about 100. In other embodiments, a chemical linker is used to connect synthetically or recombinantly produced recognition and labeling domain subsequences. Such flexible linkers are known to persons of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofinctional linkages.
The detectable labels used in the assays of the present invention, which are attached to the detection reagent, can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling). An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden (1997) Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. and in Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc., Eugene, Oreg. Patents that described the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
Primary and secondary labels can include undetected elements as well as detected elements. Useful primary and secondary labels in the present invention can include spectral labels such as green fluorescent protein, fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green(trademark), rhodamine and derivatives (e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA, CyDyes(trademark), and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, 32P, 33P, etc.), enzymes (e.g., horse radish peroxidase, alkaline phosphatase etc.), spectral colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads. The label can be coupled directly or indirectly to a component of the detection assay (e.g., the detection reagent) according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
Preferred labels include those that use: 1) chemiluminescence (using horseradish peroxidase and/or alkaline phosphatase with substrates that produce photons as breakdown products as described above) with kits being available, e.g., from Molecular Probes, Amersham, Boehringer-Mannheim, and Life Technologies/Gibco BRL; 2) color production (using both horseradish peroxidase and/or alkaline phosphatase with substrates that produce a colored product (kits available from Life Technologies/Gibco BRL, and Boehringer-Mannheim)); 3) fluorescence using, e.g., an enzyme such as alkaline phosphatase, together with the substrate AttoPhos (Amersham) or other substrates that produce fluorescent products, 4) fluorescence (e.g., using Cy-5 (Amersham), fluorescein, and other fluorescent tags); 5) radioactivity. Other methods for labeling and detection will be readily apparent to one skilled in the art.
For use of the present invention outside the laboratory, preferred labels are non-radioactive and readily detected without the necessity of sophisticated instrumentation. Preferably, detection of the labels will yield a visible signal that is immediately discernable upon visual inspection. One preferred example of detectable secondary labeling strategies uses an antibody that recognizes SAP in which the antibody is linked to an enzyme (typically by recombinant or covalent chemical bonding). The antibody is detected when the enzyme reacts with its substrate, producing a detectable product. Preferred enzymes that can be conjugated to detection reagents of the invention include, e.g., xcex2-galactosidase, luciferase, horse radish peroxidase, and alkaline phosphatase. The chemiluminescent substrate for luciferase is luciferin. One embodiment of a fluorescent substrate for xcex2-galactosidase is 4-methylumbelliferyl-xcex2-D-galactoside. Embodiments of alkaline phosphatase substrates include p-nitrophenyl phosphate (pNPP), which is detected with a spectrophotometer; 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazoliuum (BCIP/NBT) and fast red/napthol AS-TR phosphate, which are detected visually; and 4-methoxy-4-(3-phosphonophenyl) spiro[1,2-dioxetane-3,2xe2x80x2-adamantane], which is detected with a luminometer. Embodiments of horse radish peroxidase substrates include 2,2xe2x80x2azino-bis(3-ethylbenzthiazoline-6 sulfonic acid) (ABTS), 5-aminosalicylic acid (5 AS), o-dianisidine, and o-phenylenediarnine (OPD), which are detected with a spectrophotometer; and 3,3,5,5xe2x80x2-tetramethylbenzidine (TMB), 3,3xe2x80x2diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4C1N, which are detected visually. Other suitable substrates are known to those skilled in the art. The enzyme-substrate reaction and product detection are performed according to standard procedures known to those skilled in the art and kits for performing enzyme immunoassays are available as described above.
The presence of a label can be detected by inspection, or a detector which monitors a particular probe or probe combination can be used to detect the detection reagent label. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.
B. B. anthracis Protein Surface Array Protein (SAP) Nucleic Acids and Polypeptides
The binding reagents used in the assays and kits of the invention are generally obtained using a B. anthracis SAP polypeptide as an immunogen. The entire SAP can be used, or polypeptide subfragments that include an immunogenic epitope can be used. Suitable SAP immunogens can be isolated from B. anthracis cultures, or more preferably can be produced using recombinant methods.
1. SAP Polypeptides
SAP polypeptides can be produced by methods known to those of skill in the art. The amino acid sequence of a B. anthracis SAP polypeptide is provided as SEQ ID NO:1. A B. anthracis SAP polypeptide from a different strain is described in Etienne-Toumelin et al., J. Bacteriol. 177:614-620 (1995).
In a presently preferred embodiment, the SAP proteins, or immunogenic subsequences thereof, are synthesized using recombinant DNA methodology. Generally this involves creating a DNA sequence that encodes the polypeptide, modified as desired, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein.
SAP polypeptides can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeasts, filamentous fungi, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. Techniques for gene expression in microorganisms are described in, for example, Smith, Gene Expression in Recombinant Microorganisms (Bioprocess Technology, Vol. 22), Marcel Dekker, 1994. Examples of bacteria that are useful for expression include, but are not limited to, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus. Filamentous fungi that are useful as expression hosts include, for example, the following genera: Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Mucor, Cochliobolus, and Pyricularia. See, e.g., U.S. Pat. No. 5,679,543 and Stahl and Tudzynski, Eds., Molecular Biology in Filamentous Fungi, John Wiley and Sons, 1992. Synthesis of heterologous proteins in yeast is well known and described in the literature. Methods in Yeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory, (1982) is a well recognized work describing the various methods available to produce the enzymes in yeast.
SAP proteins, whether recombinantly or naturally produced, can be purified, either partially or substantially to homogeneity, according to standard procedures of the art, such as, for example, ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)). Once purified, partially or to homogeneity as desired, the polypeptides can then be used (e.g., as immunogens for antibody production).
One of skill in the art would recognize that after chemical synthesis, biological expression, or purification, the SAP protein(s) may possess a conformation substantially different than the native conformations of the constituent polypeptides. In this case, it may be necessary or desirable to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art (See, Debinski et al. (1993) J. Biol. Chem. 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem. 4: 581-585; and Buchner et al. (1992) Anal. Biochem. 205: 263-270). Debinski et al., for example, describe the denaturation and reduction of inclusion body proteins in guanidine-DTE. The protein is then refolded in a redox buffer containing oxidized glutathione and L-arginine.
One of skill also would recognize that modifications can be made to the SAP polypeptides without diminishing their immunogenic activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the polypeptide into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
2. B. anthracis SAP-encoding Nucleic Acids
Nucleic acids that encode B. anthracis are useful for the recombinant production of SAP. Such nucleic acids can be isolated, for example, by routine cloning methods. The cDNA sequence provided in SEQ ID NO:2 can be used to provide probes that specifically hybridize to a SAP gene, to a SAP mRNA, or to a SAP cDNA in a cDNA library (e.g., in a Southern or Northern blot). Once the target SAP nucleic acid is identified, it can be isolated according to standard methods known to those of skill in the art (see, e.g., Sambrook, Berger, and Ausubel, supra.).
SAP nucleic acids also can be isolated by amplification methods such as polyrnerase chain reaction (PCR), the ligase chain reaction (LCR), the tnanscription-based amplification system (TAS), the self-sustained sequence replication system (SSR). A wide variety of cloning and in vitro amplification methodologies are well-known to persons of skill. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger, Sambrook, and Ausubel (all supra.); Cashion et al., U.S. Pat. No. 5,017,478; and Carr, European Patent No. 0,246,864. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al., eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim and Levinson (Oct. 1, 1990) CandEN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh et aL (1989) Proc. Nat""l. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin. Chem. 35: 1826; Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene, 4: 560; and Barringer et al. (1990) Gene 89: 117.
A polynucleotide that encodes a SAP polypeptide can be operably linked to appropriate expression control sequences for a particular host cell in which the polypeptide is to be expressed. Such constructs are often referred to as xe2x80x9cexpression cassettes.xe2x80x9d For E. coli, appropriate control sequences include a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences typically include a promoter which optionally includes an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences. In yeast, convenient promoters include GAL1,10 (Johnson and Davies (1984) Mol. Cell. Biol. 4:1440-1448) ADH2 (Russell et al. (1983) J. Biol. Chem. 258:2674-2682), PHO5 (EMBO J. (1982) 6:675-680), and MFxcex11 (Herskowitz and Oshima (1982) in The Molecular Biology of the Yeast Saccharomyces (eds. Strathern, Jones, and Broach) Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., pp. 181-209).
Expression cassettes are typically introduced into a vector that facilitates entry into a host cell, and maintenance of the expression cassette in the host cell. Vectors that include a polynucleotide that encodes a SAP polypeptide are provided by the invention. Such vectors often include an expression cassette that can drive expression of the SAP polypeptide. To easily obtain a vector of the invention, one can clone a polynucleotide that encodes the SAP polypeptide into a commercially or commonly available vector. A variety of common vectors suitable for this purpose are well known in the art. For cloning in bacteria, common vectors include pBR322 derived vectors such as pBLUESCRIPT(trademark), and xcex-phage derived vectors. In yeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD-2. A multicopy plasmid with selective markers such as Leu-2, URA-3, Trp-1, and His-3 is also commonly used. A number of yeast expression plasmids such as YEp6, YEp13, YEp4 can be used as expression vectors. The above-mentioned plasmids have been fuilly described in the literature (Botstein et al. (1979) Gene 8:17-24; Broach et al. (1979) Gene, 8:121-133). For a discussion of yeast expression plasmids, see, e.g., Parents, B., YEAST (1985), and Ausubel, Sambrook, and Berger, all supra). Expression in mammalian cells can be achieved using a variety of commonly available plasmids, including pSV2, pBC12BI, and p9103, as well as lytic virus vectors (e.g., vaccinia virus, adenovirus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retroviruses).
The nucleic acids that encode SAP polypeptides can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for E. coli or mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes, among others. Techniques for transforming fungi are well known in the literature and have been described, for instance, by Beggs et al. ((1978) Proc. Natl. Acad. Sci. USA 75: 1929-1933), Yelton et al. ((1984) Proc. Natl. Acad. Sci. USA 81: 1740-1747), and Russell ((1983) Nature 301: 167-169). Procedures for transforming yeast are also well known (see, e.g., Beggs (1978) Nature (London), 275:104-109; and Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA, 75:1929-1933. Transformation and infection methods for mammalian and other cells are described in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989) Molecular Cloningxe2x80x94A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1994 Supplement) (Ausubel).
C. Assay Formats
The B. anthracis detection methods of the present invention can be carried out in a wide variety of assay formats. Typically, the assay methods involve immobilization of B. anthracis SAP on a solid support, followed by detection of the inmnobilized SAP. The detectable labels can be detected directly after immobilization on the solid support, for example, or indirectly by an enzymatic or other reaction that results in a detectable change in a reactant that is present in the detection assay reaction.
1. ELISA Detection Methods of the Invention
Presently preferred assay systems for use in the kit and methods of the invention are based on the enzyme-linked immunosorbent assay (ELISA) method. General methods for ELISA are well known to those of skill in the art (see, e.g., Elder et al., J. Clin. Microbiol. 16:141 (1982); Ausubel et al., supra). Generally, antigens fixed to a solid surface are detected using antigen-specific antibodies that are detected by way of an enzymatic reaction. In a presently preferred embodiment, the ELISA method used is the xe2x80x9csandwichxe2x80x9d method wherein the antigens are bound to the solid surface via an antibody bound to the solid surface. A second antibody, typically linked to an enzyme, is then contacted to the antigen, washed, then contacted with the enzyme substrate to select binding. These and other embodiments of the ELISA method are taught in, for example, Ausubel et al. xc2xa7 11.2, supra.
To immobilize SAP on the solid support, a capture reagent that specifically binds to SAP is non-diffusively associated with the support. The capture reagents can be non-diffusively immobilized on the support either by covalent or non-covalent methods, which are known to those of skill in the art. See, e.g., Pluskal et al. (1986) BioTechniques 4: 272-283. Suitable supports include, for example, glasses, plastics, polymers, metals, metalloids, ceramics, organics, and the like. Specific examples include, but are not limited to, microtiter plates, nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, beads, and also particles, such as agarose, SEPHADEX(trademark), and the like. Assay systems for use in the methods and kits of the invention include, but are not limited to, dipstick-type devices, immunochromatographic test strips and radial partition immunoassay devices, microtiter assays and flow-through devices. Conveniently, where the solid support is a membrane, the test sample can flow through the membrane, for example, by gravity, capillary action, or under positive or negative pressure.
Once the test sample has been contacted with the solid support, the solid support is then contacted with detection reagents for SAP. The solid support can be washed prior to contact with detection reagents to remove unbound reagents and test sample components. After incubation of the detection reagents for a sufficient time to bind a substantial portion of the immobilized SAP, any unbound labeled reagents are removed by, for example, washing. The detectable label associated with the detection reagents is then detected. For example, in the case of an enzyme used as a detectable label, a substrate for the enzyme that turns a visible color upon action of the enzyme is placed in contact with the bound detection reagent. A visible color will then be observed in proportion to the amount of the specific antigen in the sample.
2. Membrane-based Detection Methods of the Invention
In some embodiments, the assay methods are carried out using a membrane-based detection system such as are described in U.S. Pat. No. 5,922,615 and EP 447154. These systems employ an apparatus that includes a porous member, such as a membrane or a filter, onto which is bound a multiplicity of capture reagents that specifically bind B. anthracis SAP. The apparatus also includes a non-absorbent member with a textured surface in communication with the lower surface of the porous member. The textured surface of the non-absorbent member can be a grooved surface (e.g., analogous to the surface of a record album) or it can be composed of channels, such that when the porous and non-absorbent members are brought into contact with one another a network of capillary channels is formed. The capillary network is formed from the contact of the porous member with the textured surface of the non-absorbent member and can be constructed either before or subsequent to the initial contacting of the porous member with a fluid.
In some embodiments, the capillary communication between the porous member and the non-absorbent member favors delaying the transferal of fluid from the porous member to the capillary network formed by the porous member and the textured surface of the non-absorbent member until the volume of the added fluid substantially exceeds the void volume of the porous member. The transferal of fluid from the porous member to the network of capillary channels formed by the porous member and the textured surface of the non-absorbent member can occur without the use of external means, such as positive external pressure or vacuum, or contact with an absorbent material.
The devices of the present invention can also include an optional member which is placed in contact with the upper surface of the porous member and may be used to partition the upper surface of the device into discrete openings. Such openings can access either the porous member or the textured surface of the non-absorbent second member. The optional member can in conjunction with the non-absorbent member compose a fluid receiving zone in which there is no intervening porous member. A fluid receiving zone constructed from the non-absorbent member and the optional member provides fluid capacity in addition to that provided by the network of capillary channels created by the contact of the porous member and the non-absorbent member. The openings in the optional member may include a first fluid opening and also an additional fluid opening. The first fluid opening functions as a portal for the introduction of the first fluid added to the device. The additional fluid opening serves as an additional portal through which additional fluids may be added to the inventive device.
To perform an assay using these devices, a volume of the test sample is added to the porous member, where the sample permeates the void volume of the porous member and thereby contacts the anchor moieties immobilized on the porous member. In a non-competitive assay, the sample to be assayed is applied to the porous member and the SAP, if present, is bound by the anchor moieties. A detection reagent for SAP is then added as an additional fluid; these bind to the complex of SAP and capture reagent. Alternatively, the detection reagent can be added to the sample prior to application of the sample to the porous member so that the binding of detection reagent to SAP occurs prior to the binding of SAP to the capture reagent. In another embodiment, the capture reagent and detection reagent are added to the sample, after which the complex of capture reagent, SAP, and detection reagent binds to a binding agent that is either combined with these reagents or is immobilized on the porous member. An additional fluid containing reagents to effect a separation of free from bound labeled reagents can be added to remove excess detection reagent, if needed.
This device is designed to provide sufficient sensitivity to measure low concentrations of SAP because one can use large amounts of sample and efficiently remove the excess of detection reagent. Indeed, the efficient separation of free from bound label achieved by the network of capillary channels of this device improves the discrimination of specific SAP-associated signal over non-specific background signal. If needed, a signal developer solution is then added to enable the label of the detection moiety to develop a detectable signal. The signal developed can then be related to the concentration of the target ligand within the sample. In a preferred embodiment, the transfer of fluid between the porous first member of the device and the network of capillary channels formed by the contact of the porous member and textured surface of the non-absorbent second member of the device is generally self-initiated at the point when the total volume of fluid added to the device exceeds the void volume of the porous member, thus obviating the need for active interaction by the user to remove excess fluid from the analyte detection zone. The point at which the fluid transfer is initiated is dependent upon the objectives of the assay. Normally, it is desirable to contact the sample with all of the zones on the porous member which contain immobilized receptor. This method enables the detection of SAP in a manner that is simple, rapid, convenient, sensitive and efficient in the use of reagents.
Competitive binding assays can also be used to detect SAP. Conveniently, these assays are performed using the described devices by adding to a sample a labeled analog of SAP. The labeled analog and SAP present in the sample compete for the binding sites of the capture reagents. Alternatively, the capture reagents can be combined with the sample and labeled analogs with subsequent immobilization of the capture reagents onto the porous member through contact with a binding agent. An additional fluid to separate the free from bound label may be added to the device, followed if needed by a signal development solution to enable detection of the label of the labeled analog which has complexed with capture reagent immobilized on the porous member. The amount of labeled SAP bound to the porous member is related to the concentration of SAP in the sample.
D. Kits for Detecting Anthrax
This invention also provides kits for the detection and/or quantification of anthrax using the methods described herein. T he kits can include a container containing one or more of the above-discussed detection reagents with or without labels, and capture reagents, either free or bound to solid supports. A suitable solid support, such as a membrane, can also be included in the kits of the invention. The kits can provide the solid supports in the form of an assay apparatus that is adapted to use in the described assay. Preferably, the kits will also include reagents used in the described assays, including reagents useful for detecting the presence of the detectable labels. Other materials useful in the performance of the assays can also be included in the kits, including test tubes, transfer pipettes, and the like. The kits can also include written instructions for the use of one or more of these reagents in any of the assays described herein.
The kits of the invention can also include an internal and/or an external control. An internal control can consist of the SAP polypeptide. The control antigen can conveniently be preattached to a capture reagent in a zone of the solid support adjacent to the zone to which the sample is applied. The external control can also consist of the SAP polypeptide. Typically, the antigen present in the external control will be at a concentration at or above the sensitivity limit of the assay means. The external control antigen can be diluted in the sample diluent and assayed in the same manner as would a biological sample. Alternatively, the external control SAP polypeptide can be added to an aliquot of an actual biological sample to determine the sensitivity of the assay. The kits of the present invention can contain materials sufficient for one assay, or can contain sufficient materials for multiple assays.
E. Test Samples
Samples to test for the presence of anthrax can be collected from any potential source of anthrax. Samples can be collected from, for example, the air, water, food, soil or other solids or liquids. In one embodiment, the methods and kits of the invention can be used to determine if terrorists have planted anthrax in a public area. In preferred embodiments, it is unknown whether the test sample contains B. anthracis. 
Air samples can be collected using, for example, a cyclonic collection device (see, e.g., Jensen et al., Am. Ind. Hyg. Assoc. J. 53:660-67 (1992); Cage et al., Ann. Allergy Asthma Immunol. 77:401-6 (1996)). Such a device can capture a volume of air, submit the air to turbulence such that any particles in the air (e.g., anthrax spores or SAP-coated particles) are deposited on a moist surface. Typically, air flowing through cyclonic tubes forms a vortex in the tube that induces high centrifugal forces on particles in the air (Anderson et al., Johns Hopkins APL Technical Digest 20(3) (1999)). The rotational forces segregate the larger particle to the outside of the tube. Variations in the tube diameter, length, taper angle and flow velocity determine particle separation size. Particles can then be captured by letting the particles slide down the tube walls into a filter bag or by washing the walls with a liquid and capturing the concentrate. The objects can then be collected and analyzed for the presence of anthrax. A variety of cyclonic devices are discussed in, e.g., Maddox et al. Monthly Microscopical J. 286-290 (1870); Fisher-Klostertman, Inc. Product Bulletin 218-C, 2900 West Broadway, Louisville, Ky.; Hering, xe2x80x9cImpactors, Cyclones, and Other Inertial and Gravitational Collectors,xe2x80x9d in Air Sampling Instruments for Evaluation of Atmospheric contaminants, 8th Ed., American Conference of Governmental Industrial Hygienists, Cincinnati, Ohio, 279-321 (1995) and; Stoutas, et al. J. Aerosol Sci. 25(7):1321-1330 (1994). Handheld air samplers can also be used to obtain samples that are tested according to the methods of the invention (see, e.g., Kenny et al., Am. Ind. Hyg. Assoc. J. 59:831-41 (1998)). Sampling of solid or liquid objects is known to those skilled in the art.
Several cyclonic collection devices are known, including conventional impactors and virtual impactors. Conventional impactors work by directing the particle-containing air through a nozzle onto a collection plate. A variation of the conventional impactor is the virtual impactor, which operates by directing the air stream from the nozzle to an opening with a restricted flow. Larger particles enter an opening which forms a virtual surface, and become entrained in a minor flow or reduced velocity, while smaller particles follow the major flow. The virtual impactor has the benefit of concentrating particle quantity from low density in the high volume flow to high density in the low volume flow. See, e.g., Anderson et al., supra.
A significant advantage of the assay methods and kits of the invention is that the sensitivity is such that a sample need not be cultured prior to assay. This not only provides a faster and less expensive assay, but also makes it possible to obtain a result in the field. Samples need not be sent to a laboratory facility for processing. This is particularly advantageous in military situations, in which suitable laboratory facilities may not be available.